Methods and systems for using encapsulated microbubbles to process biological samples

ABSTRACT

Methods and systems for using encapsulated microbubbles to process biological samples are disclosed. According to one aspect, a method for using encapsulated microbubbles to process a biological sample includes creating a mixture comprising encapsulated microbubbles mixed with a biological sample and adding activation energy to the mixture to cause at least some of the microbubbles to oscillate or burst and thereby process the sample, including effecting cell lysis, shearing DNA, and/or performing tissue dispersion.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.15/990,171, filed May 25, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/432,747, filed Mar. 31, 2015, and which issuedas U.S. Pat. No. 9,982,290 on May 29, 2018, which is a National StageEntry of International Patent Application No. PCT/US2013/063397, filedOct. 4, 2013, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/709,488, filed Oct. 4, 2012.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NumbersEB008733 and EB009066 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The subject matter described herein relates to methods and systems forprocessing biological samples. More particularly, the subject matterdescribed herein relates to methods and systems for using encapsulatedmicrobubbles to process biological samples.

BACKGROUND

The transformation of a normal cell into a cancer cell is the result ofan accumulation of genetic mutations or epigenetic changes that lead toaberrant gene expression. Understanding where and when these changesoccur in any given tumor type is a primary focus of cancer research.Since cellular transformation is often unique for each patient, adiagnostic test that provides a customized genome-based profile of atumor is very desirable and is likely be a standard in the future ofoncological medicine. Genetic mutations in transformed cells are nowvery commonly profiled using next-generation sequencing (NGS).Incorporation of NGS technology into routine clinical diagnostics,however, will require overcoming a number of hurdles includingstreamlining of sample processing.

Random, unbiased fragmentation of DNA is necessary for NGS and is a keystep in building any genomic library for sequencing. When NGS is used todetermine genetic changes, the desired length of each DNA fragment inbase pairs (bp) depends on the maximum possible read length of thesequencer. If fragments exceed the maximum length, they will beincompletely read. If they are too small, or degraded, then they will beexcluded from the read. Therefore, consistent DNA fragmentation isrequired for quality NGS data. DNA shearing is also used in processessuch as chromatin immunoprecipitation (ChIP), formaldehyde-assistedinterrogation of regulatory elements (FAIRE), and DNA sequencing. Foreach of these techniques, it is important that DNA is sheared to aconsistent size in the shortest time possible. Chromatin from cells thathave been subjected to formaldehyde crosslinking (e.g., from the ChIPand FAIRE techniques), are particularly resistant to conventional DNAshearing techniques. Thus, the time required to shear formaldehydecross-linked DNA makes it difficult to integrate this step intoautomated protocols with large sample sets.

Conventional methods for fragmentation of DNA include enzymaticdigestion, sonication, nebulization, and hydrodynamic shearing. Whileall of these techniques are widely used, each has advantages anddisadvantages. Enzymatic digestion using DNase I, MNase, or restrictionenzymes is very efficient, but introduces an enzyme bias. Regions oftranscriptionally silent, tightly packed (heterochromatic) DNA and DNAwith high G-C content can be refractive to enzymatic digestion and manyenzymes only create nicks in the DNA instead of cutting completelythough both strands. The nebulization process shears solubilized DNA byforcing it through a pressurized nozzle (atomization). This method isfast, but requires large quantities of DNA and often results in a largedistribution in the DNA fragment size and cross-contamination betweensamples. Hydrodynamic shearing involves forcing solubilized DNA througha mesh. It has the advantage of rapidly producing small DNA fragments ofnearly uniform length. This method, however, is costly and the screenused for shearing is prone to clogging and cross-contamination betweensamples. Similar to enzymatic digestion, heterochromatic DNA or DNA withhigh G-C content are very difficult to shear, which creates a biastoward better shearing efficiency in euchromatic and A-T rich regions.Although NGS involves fragmentation of purified genomic DNA, ChIPrequires fragmentation of DNA from the nuclei of intact, formaldehydecross-linked cells. Formaldehyde crosslinks protein to DNA, and as aresult cells are very rigid and particularly resistant to lysis.Therefore, DNA fragmentation for fixed samples such as ChIP or FFPEtissue is even more challenging. The summary is that current DNAfragmentation methods are a bottleneck for diagnostic assays such as NGSand ChIP, and that a substantial improvement in methods for DNAfragmentation would have a significant impact.

Presently, DNA fragmentation in academic laboratories is commonlyperformed using a probe-based or acoustic sonicator. Sonication usesuncontrolled cavitation to shear DNA. Conventional DNA sonicators rangefrom a single probe to multi-sample acoustic water bath sonicators. Themethod typically produces inconsistent results and is time consuming,thereby limiting its utility. DNA extracted from cells or tissue must beoptimized each time to ensure that fragmentation occurs to the desiredsize range. In the case of formaldehyde cross-linked samples, checkingDNA fragment size involves an overnight incubation, so optimization cantake several days for each sample type. Therefore, an inexpensive methodthat provides shearing consistency independent of cell or tissue typeand that can be performed rapidly would be very valuable and would havea significant impact on the use of technologies like NGS as a companiondiagnostic for cancer.

There are few methods currently available to increase DNA fragmentationefficiency by an acoustic or probe sonicator. Borosilicate glass beadsare sometimes added to the DNA mix during sonication, but these beadsprovide limited improvement and will reduce the lifetime of a probesonicator by causing pitting in the probe. Another technique uses a vialthat contains a rod that enucleates bubbles during sonication.Cavitation nucleated from this rod help to shear DNA. While thesemicrobubble nucleating rods improve consistency results in thesonicator, they release plastic residue that can clog columns indownstream applications, and are very costly (five dollars per sample).Thus, current DNA fragmentation methods are a bottleneck for thecreation of NGS libraries.

Accordingly, in light of these disadvantages associated withconventional techniques for DNA shearing, there exists a need for moreconsistent and efficient techniques for DNA shearing. More specifically,there exists a need for methods and systems for using encapsulatedmicrobubbles to process biological samples.

SUMMARY

According to one aspect, the subject matter described herein includes amethod for using encapsulated microbubbles to process a biologicalsample according to an embodiment of the subject matter describedherein. The method includes creating a mixture comprising encapsulatedmicrobubbles mixed with a biological sample and adding activation energyto the mixture to cause at least some of the microbubbles to oscillateor burst and thereby process the sample.

According to another aspect, the subject matter described hereinincludes a system for delivering a solution of microbubbles to abiological sample according to an embodiment of the subject matterdescribed herein. The system includes a container for holding thesolution of microbubbles and at least one outlet for delivering thesolution of microbubbles to a biological sample.

According to yet another aspect, the subject matter described hereinincludes a system for delivering microbubbles to a biological sampleaccording to an embodiment of the subject matter described herein. Thesystem includes a container for holding a solution that will formulateencapsulated microbubbles when processed and at least one outlet fordelivering the solution to a biological sample.

The subject matter described herein can be implemented in software incombination with hardware and/or firmware. For example, the subjectmatter described herein can be implemented in software executed by aprocessor. In one exemplary implementation, the subject matter describedherein can be implemented using a non-transitory computer readablemedium having stored thereon computer executable instructions that whenexecuted by the processor of a computer control the computer to performsteps. Exemplary computer readable media suitable for implementing thesubject matter described herein include non-transitory computer-readablemedia, such as disk memory devices, chip memory devices, programmablelogic devices, and application specific integrated circuits. Inaddition, a computer readable medium that implements the subject matterdescribed herein may be located on a single device or computing platformor may be distributed across multiple devices or computing platforms.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the subject matter described herein will now beexplained with reference to the accompanying drawings, wherein likereference numerals represent like parts, of which:

FIG. 1 is a flow chart illustrating an exemplary process for usingencapsulated microbubbles to process a biological sample according toyet another embodiment of the subject matter described herein;

FIG. 2 is a flow chart illustrating a method for creating the biologicalsample and microbubble mixture according to an embodiment of the subjectmatter described herein;

FIG. 3 is a flow chart illustrating a method for creating the biologicalsample and microbubble mixture according to another embodiment of thesubject matter described herein;

FIG. 4 is a flow chart illustrating a method for creating the biologicalsample and microbubble mixture according to another embodiment of thesubject matter described herein;

FIGS. 5A and 5B are block diagrams illustrating exemplary systems fordelivering microbubbles to a biological sample according to embodimentsof the subject matter described herein;

FIGS. 6A through 6F are block diagrams illustrating exemplary systemsfor delivering microbubbles to a biological sample according to otherembodiments of the subject matter described herein;

FIG. 7 shows the results of DNA fragmentation in the presence andabsence of microbubbles; and

FIG. 8 shows the results of densitometry performed on three independentbiological replicates after 30 seconds sonication time.

DETAILED DESCRIPTION

In accordance with the subject matter disclosed herein, methods andsystems for using encapsulated microbubbles to process biologicalsamples are provided. The addition of controlled sized lipidmicrobubbles to an acoustic sonication process significantly improvesthe efficiency and consistency of DNA fragmentation and the presence ofthe lipid microbubbles does not interfere with downstreamfluorescence-based analyses such as quantitative PCR. This technique canbe used to greatly improve sample processing efficiency and robustness.Microbubbles may also be used to enhance other sample processes, such asto effect cell lysis, to perform tissue dispersion, and to detachtissues from tissue culture (TC) plates in preparation for transfer oftissue to polymerase chain reaction (PCR) plates.

Reference will now be made in detail to exemplary embodiments of thepresent invention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

Microbubbles, which are bubbles that are typically in the 1-10 microndiameter size range, have been used in medical diagnostics as contrastagents for ultrasound imaging for approximately two decades. For medicalapplications, these microbubbles utilize an encapsulating shell,typically a lipid, and high molecular weight gas core to maximizestability. The highly compressible core of a gas filled microbubbleenables it to compress and expand in a pressure field. In an acousticfield, this oscillation happens at the frequency of the sound wave. Formedical acoustics, this is on the order of megahertz (MHz). It has beenshown through the use of high-speed photography that expansion andcompression velocity of microbubbles in an acoustic field can be on theorder of 350 meters per second even at only moderate acoustic pressures(2.4 MHz, 1.2 megapascals (MPa)). When the bubble remains intact, thisphenomenon is called stable cavitation. At higher acoustic energies,these microbubbles can oscillate to such a violent extent that thebubbles will break up in a process called transient cavitation.

Cavitation in general is a very mechanically vigorous process, and theintensity typically increases as acoustic pressure is increased andacoustic center frequency is decreased. At sufficient acousticparameters microbubble-mediated cavitation is well known to causevarious effects such as the disruption of cell membranes, the breakup upof blood clots, and the permeabilization of vascular endothelium. Theexact mechanism is still poorly understood, however. Furthermore,because microbubbles are orders of magnitude larger in size than DNAbase pairs, it was not known whether microbubbles would perform as aneffective reagent for DNA fragmentation in an acoustic sonicator. Sincethe microbubbles consist of a lipid layer surrounding an inert gas, andthey were used in small quantities (10 micrograms (μg) per sample), theywere not expected to interfere with downstream analyses such as thefluorescent-based quantitative polymerase chain reaction (qPCR), NGS, orChIP, however, and were therefore a good candidate for study.

FIG. 1 is a flow chart illustrating an exemplary process for usingencapsulated microbubbles to process a biological sample according toyet another embodiment of the subject matter described herein. In theembodiment illustrated in FIG. 1 , step 100 includes creating a mixturethat contains encapsulated microbubbles mixed with a biological sample,and step 102 includes adding activation energy to the mixture to causeat least some of the microbubbles to oscillate or burst, therebyprocessing the sample. In one embodiment, a majority of the microbubbleshave a diameter in the range from 0.1 microns to 10 microns.

The inclusion of microbubbles into a biological sample and theirsubsequent activation has been found to increase the efficiency of anumber of processes. For example, the biological sample may include DNAor DNA that has been cross-linked to protein, in which case processingthe sample may include shearing the DNA. The biological sample mayinclude cells, including but not limited to bacteria or yeast cells,where processing the sample may include effecting cell lysis. Thebiological sample may include tissue, in which case processing thesample includes performing tissue dispersion. The method may besuccessfully applied to a variety of types of tissues, including freshtissue, cryogenically preserved tissue, and fixed and paraffin embeddedtissue.

There are a number of ways in which activation energy may be added tothe mixture, including but not limited to sonicating the mixture,exposing the mixture to laser light, and exposing the mixture to heat.Sonicating the mixture may include applying sonic energy in the 0.01 MHzto 10.0 MHz frequency range.

There are a number of ways to create the biological sample andmicrobubble mixture. Some of these are illustrated in FIGS. 2, 3, and 4.

FIG. 2 is a flow chart illustrating a method for creating the biologicalsample and microbubble mixture according to an embodiment of the subjectmatter described herein. In the embodiment illustrated in FIG. 2 , thebiological sample and microbubble mixture is created by adding asolution of encapsulated microbubbles to the sample (step 200) andmixing them together (step 202.) The solution of encapsulatedmicrobubbles may be pre-prepared, or may be prepared by mixingdehydrated microbubbles with a hydrating solution that re-suspends themicrobubbles and mixing the resulting solution of encapsulatedmicrobubbles with the biological sample.

FIG. 3 is a flow chart illustrating a method for creating the biologicalsample and microbubble mixture according to another embodiment of thesubject matter described herein. In the embodiment illustrated in FIG. 3, the biological sample and microbubble mixture is created by adding asolution of encapsulated droplets, which may be nanodroplets, to thesample (step 300), mixing the solution with the sample (step 302), andconverting at least some of the droplets to encapsulated microbubbles bythe addition of conversion energy (step 303.) In one embodiment, thesenanodroplets are identical in composition to the microbubbles, yetremain metastable in liquid form until the addition of acoustic energy,after which they vaporize into microbubbles.

Both standard microbubbles and vaporized phase change nanodroplets wouldhave identical acoustically active properties once in the gas form. Thebenefit of the nanodroplets is that they can be prepared in sub-micronform, such as 100 to 750 nanometers, small sizes which makes them nearlyneutrally buoyant and more likely to impregnate intracellular spaces forenhanced dispersion. Once vaporized, they grow in diameter byapproximately a factor of 6 due to the liquid to gas volume change. Theresulting bubble could play a significant role in tissue dispersion,with the additional advantage of enhanced penetration of tissue while inthe liquid nanodroplet precursor stage compared to the larger and morebuoyant microbubbles. The phase-change droplets have the advantage thatthey sink rather than float in solution, which might increaseinteraction with materials at the bottom of a sample chamber.

Liquid perfluorocarbon-based micro- and nano-droplets may be createdwith the same type of solution that is used for lipid-coatedmicrobubbles. Microbubbles are mechanically agitated in a 3 mL vial toform a polydisperse size distribution. Due to the low boiling point ofthe perfluorocarbon gas, transition to the liquid phase is achievable.By increasing the pressure on the headspace of the sealed vial using acustom apparatus, the gas core will transition from the gas phase to theliquid phase, thus creating a polydisperse distribution of lipid-coatedperfluorocarbon-based micro- and nano-droplets. By supplying enoughacoustic energy, the droplets may transition back to the gas phase andsubsequently cavitate to provide the necessary mechanism to expedite thelysis of cells.

The droplets may be converted to microbubbles before the solution isadded to the biological tissue, or they may not be converted until afterthe solution is added to the biological tissue, which providesadditional benefits in some circumstances. For example, the conversionof droplets to microbubbles after the droplets have been added to thebiological tissue can effect cell lysis and can help detach tissues froma tissue culture plate in preparation for transfer to PCR plates, whichmay help avoid a scraping step.

In one embodiment, the droplets may include a shell surrounding a liquidcore which converts to a gas upon the addition of acoustic, thermal, oroptical energy. The liquid core may include a hydrocarbon or aperfluorocarbon. Examples of liquids include but are not limited toisopentane, perfluoropentane, perfluorohexane, perfluorobutane, andperfluoropropane.

Conversion energy may be added using a number of techniques, includingsonicating the mixture, exposing the mixture to laser light, etc. In oneembodiment, for example, the droplets may be converted into microbubblesby sonicating the mixture using ultrasound in the 0.01 MHz to 10.0 MHzfrequency range.

FIG. 4 is a flow chart illustrating a method for creating the biologicalsample and microbubble mixture according to another embodiment of thesubject matter described herein. In the embodiment illustrated in FIG. 4, the biological sample and microbubble mixture is created by adding tothe biological sample a solution containing reagents that will formulateencapsulated microbubbles upon the addition of energy (step 400), thenadding formation energy to a biological sample to induce the formationof encapsulated microbubbles in the biological sample (step 402.)

In one embodiment, the reagent solution may be added to the biologicalsample and the resulting mixture is subjected to the formulation energy,which creates the microbubbles. In one example, the reagent solution maybe mixed with the biological sample and placed into a test tube or othercontainer that contains air or some other gas. The test tube is thensealed and subjected to vigorous shaking, which creates themicrobubbles. A surfactant, emulsifier, polymer, or protein may be addedto the sample prior to or during the addition of the formation energy toenhance bubble stability so that the microbubbles persist until theaddition of the activation energy. In another example, nanodroplets maybe added to the biological sample, and the resulting mixture may besubjected to sonication or other form of formation energy, which causesthe nanodroplets to vaporize into microbubbles. Sonication may includeusing ultrasound having a frequency in the range from 0.01 MHz to 10.0MHz. Other types of formation energy include light, such as laser light,and heat.

All of the steps described above may be applied in parallel for thepurpose of performing high throughput processing of multiple biologicalsamples. For example, adding the solution of encapsulated microbubblesor encapsulated droplets to a sample may involve adding the solutionmultiple biological samples located in individual wells of a multi-wellsample plate and mixing the solution with the samples in the wells.Likewise, adding the formation or activation energies may involve addingthe formation or activation energies to multiple biological sampleslocated in individual wells of a multi-well sample plate.

FIG. 5A illustrates a system for delivering microbubbles to a biologicalsample according to an embodiment of the subject matter describedherein. In the embodiment illustrated in FIG. 5A, system 500 includes acontainer 502 for holding a solution of microbubbles, and at least oneoutlet 504 for delivering the solution of microbubbles to a biologicalsample 506. In the embodiment illustrated in FIG. 5A, biological sample506 is contained in one or more wells of a multi-well plate 508. In oneembodiment, container 502 may have multiple outlets 504 for deliveringthe solution of microbubbles to different wells of multi-well plate 508in parallel for high throughput processing. In other embodiments, sample506 may be contained in a single well plate, a test tube, or othercontainer. In one embodiment, the solution of microbubbles may be mixedwith the sample 506 in a mixing step.

In one embodiment, system 500 may include an energy source 510 forproviding activation energy to the microbubbles within the sample(s)such that the microbubbles oscillate or burst and thereby process thesample. Examples of activation energy include, but are not limited to,thermal energy, sonic or ultrasonic energy, and light energy. In oneembodiment, for example, activation energy may be provided usingultrasound having a frequency in the range from 0.01 MHz to 10 MHz.

System 500 may be used to perform different processes, which may beperformed on different types of samples. The cavitation caused byoscillating or rupturing microbubbles can detach biological samples froma TC plate in preparation for transferal of the sample to a PCR, forexample. In one embodiment, sample 506 may include DNA or DNA that hasbeen cross-linked to protein, in which case processing sample 506 mayinclude shearing the DNA. In another embodiment, sample 506 may includecells, such as (but not limited to) bacteria and yeast cells, in whichcase processing sample 506 may include effecting cell lysis. In anotherembodiment, sample 506 may include tissue, in which case processingsample 506 may include performing tissue dispersion. System 500 may beused to process various kinds of tissue, including fresh tissue,cryogenically preserved tissue, and fixed and paraffin embedded tissue.In one embodiment, microbubbles having a diameter in the range from 0.1microns to 10 microns may be used for DNA shearing or other processes.

FIG. 5B illustrates a variation of system 500. In the embodiment shownin FIG. 5B, system 500 may also include a second container 512 forholding dehydrated microbubbles, which are mixed with a solution 514 andthus re-suspended before being provided to container 502.

FIG. 6A illustrates a system for delivering microbubbles to a biologicalsample according to an embodiment of the subject matter describedherein. In the embodiment illustrated in FIG. 6A, system 600 includes acontainer 602 for holding a solution that will formulate encapsulatedmicrobubbles when processed, and at least one outlet 604 for deliveringthe solution to a biological sample 606. In one embodiment, container602 may have multiple outlets 604 for delivering the solution ofmicrobubbles to different wells of multi-well plate 608 in parallel forhigh throughput processing. In other embodiments, sample 606 may becontained in a single well plate, a test tube, or other container. Inone embodiment, the solution of microbubbles may be mixed with thesample 606 in a mixing step. In one embodiment, system 600 may includean energy source 610 for providing activation energy to the microbubbleswithin the sample(s) such that the microbubbles oscillate or burst andthereby process the sample. Examples of activation energy include, butare not limited to, thermal energy, sonic or ultrasonic energy, andlight energy. In one embodiment, for example, activation energy may beprovided using ultrasound having a frequency in the range from 0.01 MHzto 10 MHz.

Like system 500, system 600 may be used to perform different processes,which may be performed on different types of samples. The cavitationcaused by oscillating or rupturing microbubbles can detach biologicalsamples from a TC plate in preparation for transferal of the sample to aPCR, for example. In one embodiment, sample 606 may include DNA or DNAthat has been cross-linked to protein, in which case processing sample606 may include shearing the DNA. In another embodiment, sample 606 mayinclude cells, such as (but not limited to) bacteria and yeast cells, inwhich case processing sample 606 may include effecting cell lysis. Inanother embodiment, sample 606 may include tissue, in which caseprocessing sample 606 may include performing tissue dispersion. System600 may be used to process various kinds of tissue, including freshtissue, cryogenically preserved tissue, and fixed and paraffin embeddedtissue. In one embodiment, microbubbles having a diameter in the rangefrom 0.1 microns to 10 microns may be used for DNA shearing or otherprocesses.

Unlike system 500, however, system 600 includes a microbubble-formingapparatus 612 for processing the solution to form encapsulatedmicrobubbles within the solution. As will be described in more detailbelow, in one embodiment the encapsulated microbubbles are formed priorto providing the solution to sample 606 and in another embodiment theencapsulated microbubbles are formed after the solution has beenprovided to sample 606.

FIG. 6B illustrates a variation of system 600. In the embodimentillustrated in FIG. 6B, microbubble forming apparatus 612 includes aprobe for adding sonic energy to the solution in the presence of gas toinduce the formation of the microbubbles in the solution before it isadded to the biological sample.

FIG. 6C illustrates another variation of system 600. In the embodimentillustrated in FIG. 6C, microbubble forming apparatus 612 includes amechanical mixer or impeller that rapidly agitates the solution in thepresence of gas, forming the microbubbles in the solution, before thesolution is added to the biological sample.

FIG. 6D illustrates yet another variation of system 600. In theembodiment illustrated in FIG. 6D, microbubble forming apparatus 612includes one or more fluid systems that force gas surrounded by liquidthrough an opening or that force gas through a porous membrane, whichproduces microbubbles.

In one embodiment, the solution that will formulate encapsulatedmicrobubbles when processed includes encapsulated liquid droplets ornanodroplets. In one embodiment, the droplets comprise a shellsurrounding a liquid core which converts to a gas upon the addition ofacoustic, thermal, or optical energy. The liquid core can include, butis not limited to including, a hydrocarbon or perfluorocarbon. In oneembodiment, the liquid core includes isopentane, perfluoropentane,perfluorohexane, perfluorobutane, and/or perfluoropropane. In oneembodiment, some or all of the droplets have a diameter in the rangefrom 100 nanometers to 750 nanometers.

FIGS. 6E and 6F illustrate yet other variations of system 600. In theembodiments illustrated in FIG. 6E and 6F, container 602 holds asolution that contains nanodroplets, and system 600 includes an energysource 613 for converting the droplets into microbubbles. In FIG. 6E,energy source 613 converts the droplets into microbubbles prior tomixing the resulting solution with biological sample 606.

In FIG. 6F, energy source 613 converts the droplets into microbubblesafter the droplets and solution have been mixed with biological sample606. In this manner, the expansion of the droplet into a microbubble inresponse to being subjected to the conversion energy provided by energysource 612 may be used to preprocess sample 606 prior to the furtherprocessing that will occur when activation energy is added to themicrobubbles so created. In these embodiments, energy source 613 may bethe same as or different from energy source 610, depending on what typesof energy are being used for conversion and activation, respectively. Inany of the embodiments illustrated herein, the solution being suppliedto the biological sample may include other substances that maycontribute to the creation of or stabilization of droplets or bubbles,including but not limited to surfactants, emulsifiers, polymers, orproteins.

Specific Embodiments

In one embodiment, lipid monolayer-coated microbubbles were createdusing 1,2-distearoyl-snglycero-3-phosphocholine (DSPC)(Avanti PolarLipids, Alabaster, Ala.) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene-glycol)- 2000 (DSPE-PEG2000) (Avanti Polar Lipids,Alabaster, Ala.) in a 9 to 1 molar ratio as previously described. Thelipids were dissolved in a buffer solution comprised ofphosphate-buffered saline (PBS), propylene glycol, and glycerol (16:3:1)for a total lipid concentration of 1.0 mg/mL. The resulting lipidsolution was placed into 3 mL glass vials in 1.5 mL aliquots. The vialswere sealed with rubber septa and 5 capped. Finally, the air in the vialheadspace was removed via a custom vacuum apparatus and replaced withdecafluorobutane (Fluoromed, Round Rock, Tex.). The vial was shakenvigorously for 45 seconds using a high-speed mixer (Vialmix,Bristol-Myers Squibb Medical Imaging, North Billerica, Mass.) to producea polydisperse distribution (Mean Diameter: 1.07±0.9, Concentration:1.0×1010 #/mL) of lipid-coated microbubbles.

Sonication was performed in a Covaris E110 sonicator in a 96-wellpolypropylene plate (Thermo Scientific, AB-0900). Genomic DNA (gDNA) wasextracted from HEK293T cells and distributed at 10 μg per well in afinal volume of 150 microliters (uL) in 10 mM Tris-HCl, pH 8.0. Ourtarget fragment range was 300-500 base pairs (bp). Ten μL of the 1 mg/mLmicrobubble suspension was added to the appropriate wells. Fragmentationwas confirmed by running 1 μg of DNA on a 2% agarose gel containing SYBRGreen and visualizing on a Typhoon Trio+imager.

FIG. 7 shows the results of DNA fragmentation in the presence andabsence of microbubbles. Ten micrograms of DNA purified from humanHEK293T cells was resuspended in 150 uL final volume per well of a96-well plate and sonicated in a Covaris E110 sonicator for 30 seconds(Lanes 3-8) or 1 minute (Lanes 9-14) in triplicate. DNA fragmentationwithout any additive is inefficient (Lanes 3-5 and 9-11). Addition of 10uL of one micron lipid encapsulated microbubbles significantly increasedthe efficiency of fragmentation (Lanes 6-8 and 12-14). DNA ladders are:1 Kb plus (Fermentas, Lane 1), and 100 bp (Invitrogen, Lane 2). Thisimage represents one of three biological replicates. The samples withmicrobubbles were efficiently sheared to the desired 300-500 bp rangewithin 30 seconds (Lanes 6-8), while the samples without microbubblesshowed very little fragmentation (Lanes 3-5 and 6-8).

FIG. 8 shows the results of densitometry performed on three independentbiological replicates after 30 seconds sonication time. Densitometryanalysis indicates that DNA fragmentation is centered in the 200-500 bprange for samples sonicated for 30 seconds in the presence oflipid-encapsulated microbubbles. Percent band density was calculatedusing the rolling ball method (ImageQuant TL, GE) by dividing thedensity for the indicated size range by the density for the entire lane.Error bars represent the standard deviation for 3 biological replicateswith 3 technical replicates each. The addition of microbubble reagent tothe acoustic sonication process significantly improved the consistencyof DNA fragmentation by producing fragments within a narrow sizedistribution.

Analysis to assess DNA integrity confirmed that the presence ofmicrobubbles does not interfere with downstream fluorescent-basedanalyses. Three biological replicates of gDNA fragmented in the presenceof microbubbles from FIG. 7 were removed from the sonication vessel anddirectly used for qPCR using a SYBR green master mix and the absolutequantification method with primers representing various genomic loci.When compared to a standard curve of unsonicated human genomic DNA,10-fold serial dilutions of fragmented DNA could be linearly amplified.The PCR products were subjected to dissociation curve analysis toconfirm that only a single PCR product was present. Both the standards,and the microbubble fragmented DNA produced a single amplificationproduct. In short, it was confirmed that the presence of lipidmicrobubbles in the fragmented DNA does not interfere with SYBRgreen-based qPCR analysis.

Overall, the addition of microbubbles to the DNA fragmentation processis an innovative approach that allows purified gDNA to be consistentlyfragmented in a manner of seconds. The same techniques may be applied toextraction and fragmentation of DNA from formalin fixed paraffinembedded (FFPE) tissue slices and cryopreserved biopsy samples. Theshort sonication times that are possible with microbubble addition couldgreatly improve sample integrity by decreasing sample handling whileincreasing processing efficiency and robustness.

An Example DNA Shearing Protocol:

-   -   1. Grow human cell line on 10 cm plates (˜1-3×10^ 6 cells per        plate).    -   2. For formaldehyde fixed cells, fix cells with 1% formaldehyde        at room temperature with gentle shaking for 5 minutes. For        non-fixed cells, proceed directly to step 4.    -   3. Stop cross-linking reaction with 125 mM glycine for 5 minutes        at room temperature with gentle shaking.    -   4. Wash cells on ice 2 times with 10 mL phosphate buffered        saline, pH 7.5 (PBS).    -   5. Scrape cells into 1 mL PBS, transfer to 1.7 mL tube, and        centrifuge at 500×g for 5 minutes. Remove supernatant.    -   6. Wash plates with 1 mL PBS, scrape again, and add to tubes        with pellets. Centrifuge at 500×μg for 5 minutes in Eppendorf        5430 table top centrifuge. Remove supernatant.    -   7. Flash-freeze pellets in liquid nitrogen. Store at −80 C until        needed. Each pellet contains ˜1×10^6 cells.    -   8. Re-suspend cell pellet in 150 uL lysis buffer (10 mM Tris, pH        8.0/100 mM NaCl/1 mM EDTA/2% Triton X-100/1% SDS) with 1×        protease inhibitor cocktail (Roche #05056489001).    -   9. Transfer 150 uL of cell suspension to one well of a 96-well        PCR plate (ABgene #SP-0410).    -   10. Add 10 uL of polydispersed mechanically agitated lipid        microbubbles to each well.    -   11. Seal plate with heat sealer.    -   12. Sonication is performed using a Covaris model E110 acoustic        sonicator. Sonicate each well for 1 minute using the following        settings: 20% duty cycle/8 intensity/200 cycles per burst.    -   13. Remove cover. Add 10 uL more of polydispersed lipid        microbubbles to each well.    -   14. Seal plate with heat sealer.    -   15. Sonicate each well for 4 minutes using the following        settings: 20% duty cycle/8 intensity/200 cycles per burst.    -   16. Centrifuge plate at 1,300×g (1,500 rpm) for 5 minutes at 4 C        to remove crude cellular debris.    -   17. Remove seal.    -   18. Transfer sonicated lysates to 1.7 mL tubes. Centrifuge at        high speed for one minute to pellet cell debris.    -   19. Transfer 10 uL from each tube to a fresh tube. Add 190 uL        TE, pH 8.0 and 2 uL 5 mg/mL RNase. Incubate tubes at 37 C for 1        hour. Add 200 uL 2× proteinase K buffer (50 mM Tris, pH 8.0/12.5        mM EDTA, pH 8.0/300 mM NaCl/1% SDS) and 2 uL 10 mg/mL        proteinase K. Incubate at 55 C overnight. Extract samples by        phenol:chloroform and confirm fragment size by gel        electrophoresis.    -   20. Use the remaining 140 uL sonicated DNA in downstream        applications.

An Example Ablation Process:

-   -   1. Removal of formaldehyde cross-linked cells from a 96-well        tissue culture plate. Once cells have been formaldehyde        cross-linked, they are particularly difficult to remove from a        surface without manual scraping. To remove these cells we will        do the following:        -   a. Add formaldehyde to 1% directly to the cell culture media            in each well of a 96-well plate for 5 minutes followed by            the addition of glycine to 125 mM final concentration to            stop crosslinking reaction.        -   b. Wash cells with PBS. Add 150 uL lysis buffer (10 mM Tris,            pH 8.0/100 mM NaCl/1 mM EDTA/2% Triton X-100/1% SDS) with 1×            protease inhibitor cocktail (Roche #05056489001).        -   c. Add 100 uL (Not sure about the exact amount) of            lipid-coated perfluorocarbon droplets. The droplets sink to            the bottom of the well where the fixed cells are located.            Seal plate.        -   d. Place plate in Covaris E110 sonicator. Sonicate for 1            minute per well.        -   e. Remove seal, transfer resuspended cells to 96-well PCR            plate (ABGene #SP-0410). Proceed with sonication as            described in Step #13 in tested protocol, above. The            droplets will “activate,” by vaporizing and subsequently            cavitate in the presence of sonication.

Other Permutations:

-   -   Use of microbubbles with polymer or protein shells    -   Use of microbubbles with different gas cores—such as oxygen,        air, or different perfluorocarbons    -   Use of phase-change droplets which convert to microbubbles upon        acoustic energy    -   Use of microbubbles with different sizes    -   Use of varying acoustic energies, duty cycles, and frequencies        to optimize interaction with microbubbles    -   Use of microfluidics systems to produce the microbubbles        directly at the site of use

Other Embodiments:

Other embodiments contemplated by the subject matter described hereininclude, but are not limited to, the following list:

-   -   1. A method for using encapsulated microbubbles to process a        biological sample, the method comprising: adding a pre-prepared        solution of encapsulated microbubbles to a biological sample;        creating a mixture by mixing the pre-prepared solution of        encapsulated microbubbles with the biological sample; and adding        energy to the mixture to cause at least some of the microbubbles        to oscillate and/or burst and thereby process the sample.    -   2. The method of list item 1 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein and        wherein processing the sample comprises shearing the DNA.    -   3. The method of list item 1 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis.    -   4. The method of list item 3 wherein the cells comprise bacteria        or yeast cells.    -   5. The method of list item 1 wherein the sample comprises        tissue, and wherein processing the sample comprises performing        tissue dispersion.    -   6. The method of list item 5 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded    -   7. The method of list item 1 wherein adding energy to the        mixture comprises sonicating the mixture    -   8. The method of list item 7 wherein sonicating the mixture        includes applying energy of a frequency between 0.01 MHz and        10.0 MHz.    -   9. The method of list item 1 wherein adding energy to the        mixture comprises exposing the sample to laser light.    -   10. The method of list item 1 wherein adding a pre-prepared        solution of encapsulated microbubbles to a simple includes        adding the solution to a plurality of biological samples located        in individual wells of a multi-well sample plate, wherein mixing        the pre-prepared solution includes mixing the solution with the        samples in the wells, and wherein adding energy to the mixtures        includes adding the energy to each of the mixtures in the wells        to perform high throughput processing of a plurality of        biological samples.    -   11. The method of list item 1 wherein a majority of the        microbubbles are between 0.1 microns and 10 microns in diameter.    -   12. A method for using encapsulated droplets to process a        biological sample, the method comprising: adding a pre-prepared        solution of encapsulated droplets to a biological sample;        creating a mixture by mixing the pre-prepared solution of        encapsulated droplets with the biological sample; and adding        energy to the mixture to convert at least some of the droplets        to gas bubbles, and adding further energy to the gas bubbles to        oscillate and/or burst the gas bubbles, and thereby process the        sample.    -   13. The method of list item 12 wherein the droplets consist of a        shell surrounding a liquid core which converts to a gas upon the        addition of acoustic, thermal, or optical energy.    -   14. The method of list item 13 wherein the liquid core includes        a hydrocarbon or a perfluorocarbon.    -   15. The system of list item 14 wherein the liquid core includes        isopentane, perfluoropentane, perfluorohexane, perfluorobutane,        or perfluoropropane.    -   16. The method of list item 12 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein, and        wherein processing the sample comprises shearing the DNA.    -   17. The method of list item 12 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis    -   18. The method of list item 17 wherein the cells comprise        bacteria or yeast cells.    -   19. The method of list item 12 wherein the sample comprises        tissue and wherein processing the sample comprises performing        tissue dispersion.    -   20. The method of list item 12 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded    -   21. The method of list item 12 wherein adding energy to the        mixture comprises sonicating the mixture.    -   22. The method of list item 21 wherein sonicating the mixture        includes adding energy having a frequency range from 0.01 MHz        and 10.0 MHz.    -   23. The method of list item 12 wherein adding energy to the        mixture comprises exposing the sample to laser light.    -   24. The method of list item 12 wherein adding a pre-prepared        solution of encapsulated droplets to a sample includes applying        the solution to a plurality of biological samples located in        individual wells of a multi-well sample plate, wherein mixing        the pre-prepared solution includes mixing the solution with the        samples in the wells and wherein adding energy to the mixture        includes adding energy to the mixtures in the wells to perform        high throughput processing of a plurality of biological samples.    -   25. The method of list item 12 where a majority of droplets are        between 0.1 and 10 microns in diameter.    -   26. A method for using encapsulated microbubbles to process a        biological sample, the method comprising: adding first energy to        a biological sample to first induce the formation of        encapsulated microbubbles in the biological sample; and adding        second energy to the sample to oscillate and/or burst the        microbubbles and thereby process the sample.    -   27. The method of list item 26 comprising adding a surfactant,        emulsifier, polymer, or protein to the biological sample prior        to or during the addition of the first energy to enhance bubble        stability so that the microbubbles persist until the addition of        the second energy.    -   28. the method of list item 26 where droplets of liquid are        added to the biological sample, prior to or during the addition        of first energy, so that said liquid droplets vaporize into        bubbles during administration of first energy, and resulting        bubbles which oscillate and/or burst in response to the addition        of the second energy.    -   29. The method of list item 26 wherein adding energy to the        sample to induce the formation of encapsulated microbubbles        includes applying laser light to the sample to form the        microbubbles.    -   30. The method of list item 26 wherein adding energy to the        sample to induce the formation of encapsulated microbubbles        includes sonicating the sample to form the microbubbles.    -   31. The method of list item 30 wherein sonicating the sample        includes applying sonic energy of a frequency 0.01 and 10.0 MHz.    -   32. The method of list item 26 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein and        wherein processing the sample comprises shearing the DNA.    -   33. The method of list item 26 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis.    -   34. The method of list item 33 wherein the cells comprise        bacteria or yeast cells.    -   35. The method of list item 26 wherein the sample comprises        tissue and wherein processing the sample comprises performing        tissue dispersion.    -   36. The method of list item 35 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded.    -   37. The method of list item 26 wherein adding the first and        second energies to a biological sample includes adding the first        and second energies to a plurality of biological samples located        in individual wells of a multi-well sample plate to perform high        throughput processing of a plurality of biological samples.    -   38. A system for delivering a solution for delivering        microbubbles to a biological sample, the system comprising: a        container for holding a solution, the solution comprising        reagents that will formulate encapsulated microbubbles upon the        addition of energy; and at least one outlet for delivering the        pre-prepared solution of to a biological sample.    -   39. The system of list item 38 comprising a probe for adding        sonic energy to the solution in the presence of gas to induce        the formation of the microbubbles in the solution before it is        added to the biological sample.    -   40. The system of list item 38 comprising a mechanical        mixer/impeller that rapidly agitates the solution in the        presence of gas, forming the microbubbles in the solution,        before the solution is added to the biological sample.    -   41. The system of list item 38 comprising at least one fluid        system which forces gas surrounded by liquid through an opening        to result in microbubble production.    -   42. The system of list item 38 comprising at least one fluid        system that forces gas through a porous membrane to form the        microbubbles.    -   43. The system of list item 38 wherein the biological sample        will then be exposed to additional thermal, sonic, or light        energy for processing.    -   44. The system of list item 43 wherein the additional energy is        ultrasound with a frequency between 0.01 to 10 MHz.    -   45. The system of list item 38 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein and        wherein processing the sample comprises shearing the DNA.    -   46. The system of list item 38 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis.    -   47. The system of list item 46 wherein the cells comprise        bacteria or yeast cells.    -   48. The system of list item 38 wherein the sample comprises        tissue and wherein processing the sample comprises performing        tissue dispersion.    -   49. The system of list item 48 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded    -   50. The system of list item 38 wherein the container includes a        plurality of outlets for delivering the solution of microbubbles        to different wells of a multi-well plate.    -   51. The system of list item 38 where the solution of reagents        includes surfactants, emulsifiers, polymers, or proteins    -   52. A system for delivering a pre-prepared solution of        microbubbles to a biological sample, the system comprising: a        container for holding the solution of microbubbles; and at least        one outlet for delivering the pre-prepared solution of        microbubbles to a biological sample.    -   53. The system of list item 52 comprising an energy source for        exposing the biological sample to thermal, sonic, or light        energy for processing    -   54. The system of list item 53 wherein the additional energy is        ultrasound with a frequency between 0.01 to 10 MHz.    -   55. The system of list item 52 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein and        wherein processing the sample comprises shearing the DNA.    -   56. The system of list item 52 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis.    -   57. The system of list item 56 wherein the cells comprise        bacteria or yeast cells.    -   58. The system of list item 52 wherein the sample comprises        tissue and wherein processing the sample comprises performing        tissue dispersion.    -   59. The system of list item 58 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded.    -   60. The system of list item 52 wherein the container includes a        plurality of outlets for delivering the solution of microbubbles        to different wells of a multi-well plate.    -   61. The system of list item 52 wherein a majority of the        microbubbles are between 0.1 and 10 microns in diameter.    -   62. A system for delivering a pre-prepared solution of        encapsulated liquid droplets to a biological sample, the system        comprising: a container for holding the solution of encapsulated        liquid droplets; and at least one outlet for delivering the        pre-prepared solution of encapsulated liquid droplets to a        biological sample.    -   63. The system of list item 62 where the droplet consists of a        shell surrounding a liquid core which converts to a gas upon the        addition of acoustic, thermal, or optical energy.    -   64. The system of list item 63 where the liquid core is a        hydrocarbon or perfluorocarbon.    -   65. The system of list item 64 wherein the liquid core comprises        isopentane, perfluoropentane, perfluorohexane, perfluorobutane,        or perfluoropropane.    -   66. The system of list item 62 comprising an energy source for        adding thermal, sonic, or light energy to the sample for        processing.    -   67. The system of list item 66 wherein the additional energy is        ultrasound with a frequency between 0.01 to 10 MHz.    -   68. The system of list item 62 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein and        wherein processing the sample comprises shearing the DNA.    -   69. The system of list item 62 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis.    -   70. The system of list item 69 wherein the cells comprise        bacteria or yeast cells.    -   71. The system of list item 62 wherein the sample comprises        tissue and wherein processing the sample comprises performing        tissue dispersion.    -   72. The method of list item 71 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded.    -   73. The system of list item 62 wherein the container includes a        plurality of outlets for delivering the solution of encapsulated        liquid droplets to different wells of a multi-well plate.    -   74. The system of list item 62 wherein a majority of        encapsulated liquid droplets are between 0.1 and 10 microns in        diameter.    -   75. A system for delivering a pre-prepared solution of        microbubbles to a biological sample, the system comprising: a        container which holds dehydrated microbubbles which are mixed        with a solution and thus resuspended; and at least one outlet        for delivering the resuspended microbubbles to a biological        sample.    -   76. The system of list item 75 wherein the biological sample        will then be exposed to additional thermal, sonic, or light        energy for processing    -   77. The system of list item 76 wherein the additional energy is        ultrasound with a frequency between 0.01 to 10 MHz.    -   78. The system of list item 75 wherein the biological sample        comprises DNA or DNA that has been cross linked to protein and        wherein processing the sample comprises shearing the DNA.    -   79. The system of list item 75 wherein the biological sample        comprises cells and wherein processing the sample comprises        effecting cell lysis.    -   80. The system of list item 75 wherein the cells comprise        bacteria or yeast cells.    -   81. The system of list item 75 wherein the sample comprises        tissue and wherein processing the sample comprises performing        tissue dispersion.    -   82. The method of list item 81 wherein the tissue is fresh, or        cryogenically preserved, or fixed and paraffin embedded.    -   83. The system of list item 75 wherein the container includes a        plurality of outlets for delivering the solution of encapsulated        liquid droplets to different wells of a multi-well plate.    -   84. The system of list item 75 wherein a majority of the        microbubbles are between 0.1 and 10 microns in diameter.

It will be understood that various details of the subject matterdescribed herein may be changed without departing from the scope of thesubject matter described herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

1. A system for delivering microbubbles to a biological sample, thesystem comprising: a container for holding a solution of microbubbles ora solution that will formulate encapsulated microbubbles when processed;and at least one outlet for delivering the solution of microbubbles to abiological sample.
 2. The system of claim 1 comprising an energy sourcefor providing activation energy to the microbubbles that causes themicrobubbles to oscillate or burst and thereby process the sample. 3.The system of claim 2 wherein the activation energy comprises thermal,sonic, or light energy, optionally wherein the activation energy isultrasound having a frequency in the range from 0.01 MHz to 10 MHz. 4.(canceled)
 5. The system of claim 2 wherein the biological samplecomprises DNA or DNA that has been cross-linked to protein and whereinprocessing the sample comprises shearing the DNA.
 6. The system of claim2 wherein the biological sample comprises cells and wherein processingthe sample comprises effecting cell lysis, optionally wherein the cellscomprise bacteria or yeast cells.
 7. (canceled)
 8. The system of 2wherein the sample comprises tissue and wherein processing the samplecomprises performing tissue dispersion.
 9. The system of claim 1 whereinthe sample comprises at least one of fresh tissue, cryogenicallypreserved tissue, and fixed and paraffin embedded tissue.
 10. The systemof claim 1 wherein the container includes a plurality of outlets fordelivering the solution of microbubbles or the solution that willformulate encapsulated microbubbles when processed to different wells ofa multi-well plate.
 11. The system of claim 1 wherein a majority of themicrobubbles have a diameter in the range from 0.1 microns to 10microns.
 12. The system of claim 1 comprising a second container forholding dehydrated microbubbles, which are mixed with a solution andthus re-suspended, and that provides the solution of microbubbles to thecontainer for holding the solution of microbubbles.
 13. (canceled) 14.The system of claim 1 comprising a microbubble forming apparatus forprocessing the solution to form encapsulated microbubbles within thesolution that will formulate encapsulated microbubbles when processed.15-24. (canceled)
 25. The system of claim 1 where the solution that willformulate encapsulated microbubbles when processed includes surfactants,emulsifiers, polymers, or proteins.
 26. The system of claim 14 whereinthe microbubble forming apparatus comprises a probe for adding sonicenergy to the solution that will formulate encapsulated microbubbleswhen processed in the presence of gas to induce the formation of themicrobubbles in the solution before it is added to the biologicalsample.
 27. The system of claim 14 wherein the microbubble formingapparatus comprises a mechanical mixer or impeller that rapidly agitatesthe solution that will formulate encapsulated microbubbles whenprocessed in the presence of gas, forming the microbubbles in thesolution, before the solution is added to the biological sample.
 28. Thesystem of claim 14 wherein the microbubble forming apparatus comprisesat least one fluid system that forces gas surrounded by liquid throughan opening to result in microbubble production.
 29. The system of claim14 wherein the microbubble forming apparatus comprises at least onefluid system that forces gas through a porous membrane to form themicrobubbles.
 30. The system of claim 1 wherein the solution that willformulate encapsulated microbubbles when processed comprisesencapsulated liquid droplets, optionally wherein a majority of thedroplets have a diameter in the range from 100 nanometers to 750nanometers.
 31. The system of claim 30 wherein the droplets comprise ashell surrounding a liquid core that converts to a gas upon the additionof acoustic, thermal, or optical energy.
 32. The system of claim 31wherein the liquid core comprises a hydrocarbon or perfluorocarbon,optionally at least one of isopentane, perfluoropentane,perfluorohexane, perfluorobutane, and perfluoropropane. 33-34.(canceled)
 35. The system of claim 30 comprising an energy source forconverting the droplets into microbubbles, optionally wherein the energysource is configured to convert the droplets into microbubbles before orafter the solution is delivered to the biological sample.
 36. (canceled)